RNA seq Workflow with DeSEQ

getwd()

[1] "/Users/baharsevgin/Desktop/Omics Data Analysis/RNAseq(DE) practical"

knitr::opts_chunk$set(echo = TRUE)

Error in loadNamespace(x) : there is no package called ‘knitr’

 if (!requireNamespace("BiocManager", quietly = TRUE))
     install.packages("BiocManager")

Warning in install.packages :
  lzma decoder corrupt data

  There is a binary version available but the source version is later:

installing the source package ‘BiocManager’

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* installing *source* package ‘BiocManager’ ...
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* DONE (BiocManager)

The downloaded source packages are in
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BiocManager::install("DESeq2", version = "3.14")

'getOption("repos")' replaces Bioconductor standard repositories, see 'help("repositories", package = "BiocManager")' for details.
Replacement repositories:
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Bioconductor version 3.14 (BiocManager 1.30.26), R 4.1.3 (2022-03-10)
Installing package(s) 'BiocVersion', 'DESeq2'
also installing the dependencies ‘fastmap’, ‘sys’, ‘bit’, ‘cachem’, ‘crayon’, ‘askpass’, ‘bitops’, ‘formatR’, ‘bit64’, ‘blob’, ‘memoise’, ‘plogr’, ‘cpp11’, ‘png’, ‘Biostrings’, ‘curl’, ‘jsonlite’, ‘mime’, ‘openssl’, ‘utf8’, ‘RCurl’, ‘GenomeInfoDbData’, ‘zlibbioc’, ‘matrixStats’, ‘lambda.r’, ‘futile.options’, ‘DBI’, ‘RSQLite’, ‘KEGGREST’, ‘XML’, ‘xtable’, ‘httr’, ‘farver’, ‘labeling’, ‘R6’, ‘viridisLite’, ‘magrittr’, ‘pillar’, ‘pkgconfig’, ‘GenomeInfoDb’, ‘XVector’, ‘MatrixGenerics’, ‘DelayedArray’, ‘futile.logger’, ‘snow’, ‘BH’, ‘AnnotationDbi’, ‘annotate’, ‘RColorBrewer’, ‘cli’, ‘glue’, ‘gtable’, ‘isoband’, ‘lifecycle’, ‘rlang’, ‘scales’, ‘tibble’, ‘vctrs’, ‘withr’, ‘S4Vectors’, ‘IRanges’, ‘GenomicRanges’, ‘SummarizedExperiment’, ‘BiocGenerics’, ‘Biobase’, ‘BiocParallel’, ‘genefilter’, ‘locfit’, ‘geneplotter’, ‘ggplot2’, ‘Rcpp’, ‘RcppArmadillo’

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installing the source packages ‘crayon’, ‘cpp11’, ‘GenomeInfoDbData’, ‘DBI’, ‘httr’, ‘labeling’, ‘R6’, ‘pillar’, ‘BH’, ‘gtable’, ‘lifecycle’, ‘scales’, ‘withr’, ‘ggplot2’

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* installing *source* package ‘crayon’ ...
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*** installing help indices
** building package indices
** installing vignettes
** testing if installed package can be loaded from temporary location
** testing if installed package can be loaded from final location
** testing if installed package keeps a record of temporary installation path
* DONE (httr)
* installing *source* package ‘pillar’ ...
** package ‘pillar’ successfully unpacked and MD5 sums checked
** using staged installation
** R
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
*** copying figures
** building package indices
** installing vignettes
** testing if installed package can be loaded from temporary location
** testing if installed package can be loaded from final location
** testing if installed package keeps a record of temporary installation path
* DONE (pillar)
* installing *source* package ‘gtable’ ...
** package ‘gtable’ successfully unpacked and MD5 sums checked
** using staged installation
** R
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
*** copying figures
** building package indices
** installing vignettes
** testing if installed package can be loaded from temporary location
** testing if installed package can be loaded from final location
** testing if installed package keeps a record of temporary installation path
* DONE (gtable)
* installing *source* package ‘scales’ ...
** package ‘scales’ successfully unpacked and MD5 sums checked
** using staged installation
** R
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
*** copying figures
** building package indices
** testing if installed package can be loaded from temporary location
** testing if installed package can be loaded from final location
** testing if installed package keeps a record of temporary installation path
* DONE (scales)
* installing *source* package ‘ggplot2’ ...
** package ‘ggplot2’ successfully unpacked and MD5 sums checked
** using staged installation
** R
** data
*** moving datasets to lazyload DB
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
*** copying figures
** building package indices
** installing vignettes
** testing if installed package can be loaded from temporary location
** testing if installed package can be loaded from final location
** testing if installed package keeps a record of temporary installation path
* DONE (ggplot2)

The downloaded source packages are in
    ‘/private/var/folders/db/_9mqvxh50pb53z2xy8z_xhjr0000gn/T/RtmpXjWyRf/downloaded_packages’
Old packages: 'askpass', 'bit', 'bit64', 'bitops', 'boot', 'cachem', 'class', 'cli', 'cluster', 'codetools', 'curl', 'farver',
  'fastmap', 'foreign', 'glue', 'jsonlite', 'KernSmooth', 'lattice', 'locfit', 'matrixStats', 'mgcv', 'mime', 'nlme', 'nnet',
  'openssl', 'Rcpp', 'RcppArmadillo', 'RCurl', 'rlang', 'rpart', 'RSQLite', 'spatial', 'survival', 'sys', 'tibble', 'utf8',
  'vctrs', 'XML'

Update all/some/none? [a/s/n]: 

a

Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/data/annotation/bin/macosx/contrib/4.1/PACKAGES.rds': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/data/annotation/bin/macosx/contrib/4.1/PACKAGES.gz': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/data/experiment/bin/macosx/contrib/4.1/PACKAGES.rds': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/data/experiment/bin/macosx/contrib/4.1/PACKAGES.gz': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/workflows/bin/macosx/contrib/4.1/PACKAGES.rds': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/workflows/bin/macosx/contrib/4.1/PACKAGES.gz': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 11573Warning: cannot open URL 'https://bioconductor.org/packages/3.14/books/bin/macosx/contrib/4.1/PACKAGES.rds': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 11573Warning: cannot open URL 'https://bioconductor.org/packages/3.14/books/bin/macosx/contrib/4.1/PACKAGES.gz': HTTP status was '404 Not Found'

  There are binary versions available but the source versions are later:

no

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/askpass_1.1.tgz'
Content type 'application/x-gzip' length 21566 bytes (21 KB)
==================================================
downloaded 21 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/bit_4.0.5.tgz'
Content type 'application/x-gzip' length 1243615 bytes (1.2 MB)
==================================================
downloaded 1.2 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/bit64_4.0.5.tgz'
Content type 'application/x-gzip' length 576409 bytes (562 KB)
==================================================
downloaded 562 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/bitops_1.0-7.tgz'
Content type 'application/x-gzip' length 29174 bytes (28 KB)
==================================================
downloaded 28 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/cachem_1.0.8.tgz'
Content type 'application/x-gzip' length 67489 bytes (65 KB)
==================================================
downloaded 65 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/class_7.3-22.tgz'
Content type 'application/x-gzip' length 96138 bytes (93 KB)
==================================================
downloaded 93 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/cli_3.6.1.tgz'
Content type 'application/x-gzip' length 1365687 bytes (1.3 MB)
==================================================
downloaded 1.3 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/cluster_2.1.4.tgz'
Content type 'application/x-gzip' length 616213 bytes (601 KB)
==================================================
downloaded 601 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/curl_5.0.0.tgz'
Content type 'application/x-gzip' length 759005 bytes (741 KB)
==================================================
downloaded 741 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/farver_2.1.1.tgz'
Content type 'application/x-gzip' length 1943005 bytes (1.9 MB)
==================================================
downloaded 1.9 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/fastmap_1.1.1.tgz'
Content type 'application/x-gzip' length 201256 bytes (196 KB)
==================================================
downloaded 196 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/foreign_0.8-84.tgz'
Content type 'application/x-gzip' length 331923 bytes (324 KB)
==================================================
downloaded 324 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/glue_1.6.2.tgz'
Content type 'application/x-gzip' length 151977 bytes (148 KB)
==================================================
downloaded 148 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/jsonlite_1.8.4.tgz'
Content type 'application/x-gzip' length 1113981 bytes (1.1 MB)
==================================================
downloaded 1.1 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/KernSmooth_2.23-21.tgz'
Content type 'application/x-gzip' length 100407 bytes (98 KB)
==================================================
downloaded 98 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/lattice_0.21-8.tgz'
Content type 'application/x-gzip' length 1349836 bytes (1.3 MB)
==================================================
downloaded 1.3 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/locfit_1.5-9.7.tgz'
Content type 'application/x-gzip' length 772384 bytes (754 KB)
==================================================
downloaded 754 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/matrixStats_0.63.0.tgz'
Content type 'application/x-gzip' length 592172 bytes (578 KB)
==================================================
downloaded 578 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/mgcv_1.8-42.tgz'
Content type 'application/x-gzip' length 3737078 bytes (3.6 MB)
==================================================
downloaded 3.6 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/mime_0.12.tgz'
Content type 'application/x-gzip' length 34812 bytes (33 KB)
==================================================
downloaded 33 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/nlme_3.1-162.tgz'
Content type 'application/x-gzip' length 2400717 bytes (2.3 MB)
==================================================
downloaded 2.3 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/nnet_7.3-19.tgz'
Content type 'application/x-gzip' length 129034 bytes (126 KB)
==================================================
downloaded 126 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/openssl_2.0.6.tgz'
Content type 'application/x-gzip' length 2886168 bytes (2.8 MB)
==================================================
downloaded 2.8 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/Rcpp_1.0.10.tgz'
Content type 'application/x-gzip' length 3242867 bytes (3.1 MB)
==================================================
downloaded 3.1 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/RcppArmadillo_0.12.2.0.0.tgz'
Content type 'application/x-gzip' length 1570686 bytes (1.5 MB)
==================================================
downloaded 1.5 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/RCurl_1.98-1.12.tgz'
Content type 'application/x-gzip' length 1089009 bytes (1.0 MB)
==================================================
downloaded 1.0 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/rlang_1.1.1.tgz'
Content type 'application/x-gzip' length 1852475 bytes (1.8 MB)
==================================================
downloaded 1.8 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/rpart_4.1.19.tgz'
Content type 'application/x-gzip' length 980040 bytes (957 KB)
==================================================
downloaded 957 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/RSQLite_2.3.1.tgz'
Content type 'application/x-gzip' length 4340278 bytes (4.1 MB)
==================================================
downloaded 4.1 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/spatial_7.3-16.tgz'
Content type 'application/x-gzip' length 157695 bytes (153 KB)
==================================================
downloaded 153 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/survival_3.5-5.tgz'
Content type 'application/x-gzip' length 6768208 bytes (6.5 MB)
==================================================
downloaded 6.5 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/sys_3.4.1.tgz'
Content type 'application/x-gzip' length 47312 bytes (46 KB)
==================================================
downloaded 46 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/tibble_3.2.1.tgz'
Content type 'application/x-gzip' length 676416 bytes (660 KB)
==================================================
downloaded 660 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/utf8_1.2.3.tgz'
Content type 'application/x-gzip' length 196912 bytes (192 KB)
==================================================
downloaded 192 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/vctrs_0.6.2.tgz'
Content type 'application/x-gzip' length 1850681 bytes (1.8 MB)
==================================================
downloaded 1.8 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/XML_3.99-0.14.tgz'
Content type 'application/x-gzip' length 1873852 bytes (1.8 MB)
==================================================
downloaded 1.8 MB

The downloaded binary packages are in
    /var/folders/db/_9mqvxh50pb53z2xy8z_xhjr0000gn/T//RtmpXjWyRf/downloaded_packages

installing the source packages ‘boot’, ‘codetools’

trying URL 'https://cran.rstudio.com/src/contrib/boot_1.3-31.tar.gz'
Content type 'application/x-gzip' length 238458 bytes (232 KB)
==================================================
downloaded 232 KB

trying URL 'https://cran.rstudio.com/src/contrib/codetools_0.2-20.tar.gz'
Content type 'application/x-gzip' length 38683 bytes (37 KB)
==================================================
downloaded 37 KB

* installing *source* package ‘boot’ ...
** package ‘boot’ successfully unpacked and MD5 sums checked
** using staged installation
** R
** data
*** moving datasets to lazyload DB
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
** building package indices
** testing if installed package can be loaded from temporary location
** testing if installed package can be loaded from final location
** testing if installed package keeps a record of temporary installation path
* DONE (boot)
* installing *source* package ‘codetools’ ...
** package ‘codetools’ successfully unpacked and MD5 sums checked
** using staged installation
** R
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
** building package indices
** testing if installed package can be loaded from temporary location
** testing if installed package can be loaded from final location
** testing if installed package keeps a record of temporary installation path
* DONE (codetools)

The downloaded source packages are in
    ‘/private/var/folders/db/_9mqvxh50pb53z2xy8z_xhjr0000gn/T/RtmpXjWyRf/downloaded_packages’

BiocManager::install(“pheatmap”)

RNA-seq Analysis

workflow: raw counts -> normalization -> unsupervised clustering (PCA) -> DE analysis -> shrinking log2 fold change -> results exploration - can add more downstream analysis as well

BiocManager::install("pheatmap")

'getOption("repos")' replaces Bioconductor standard repositories, see 'help("repositories", package = "BiocManager")' for
details.
Replacement repositories:
    CRAN: https://cran.rstudio.com/
Bioconductor version 3.14 (BiocManager 1.30.26), R 4.1.3 (2022-03-10)
Installing package(s) 'pheatmap'
Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/data/annotation/bin/macosx/contrib/4.1/PACKAGES.rds': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/data/annotation/bin/macosx/contrib/4.1/PACKAGES.gz': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/data/experiment/bin/macosx/contrib/4.1/PACKAGES.rds': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/data/experiment/bin/macosx/contrib/4.1/PACKAGES.gz': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/workflows/bin/macosx/contrib/4.1/PACKAGES.rds': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/workflows/bin/macosx/contrib/4.1/PACKAGES.gz': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 11573Warning: cannot open URL 'https://bioconductor.org/packages/3.14/books/bin/macosx/contrib/4.1/PACKAGES.rds': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 11573Warning: cannot open URL 'https://bioconductor.org/packages/3.14/books/bin/macosx/contrib/4.1/PACKAGES.gz': HTTP status was '404 Not Found'Warning: lzma decoder corrupt data

  There is a binary version available but the source version is later:

installing the source package ‘pheatmap’

trying URL 'https://cran.rstudio.com/src/contrib/pheatmap_1.0.13.tar.gz'
Content type 'application/x-gzip' length 15162 bytes (14 KB)
==================================================
downloaded 14 KB

* installing *source* package ‘pheatmap’ ...
** package ‘pheatmap’ successfully unpacked and MD5 sums checked
** using staged installation
** R
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
** building package indices
** testing if installed package can be loaded from temporary location
** testing if installed package can be loaded from final location
** testing if installed package keeps a record of temporary installation path
* DONE (pheatmap)

The downloaded source packages are in
    ‘/private/var/folders/db/_9mqvxh50pb53z2xy8z_xhjr0000gn/T/RtmpXjWyRf/downloaded_packages’
Old packages: 'askpass', 'bit', 'bit64', 'bitops', 'cachem', 'class', 'cli', 'cluster', 'curl', 'farver', 'fastmap',
  'foreign', 'glue', 'jsonlite', 'KernSmooth', 'lattice', 'locfit', 'matrixStats', 'mgcv', 'mime', 'nlme', 'nnet',
  'openssl', 'Rcpp', 'RcppArmadillo', 'RCurl', 'rlang', 'rpart', 'RSQLite', 'spatial', 'survival', 'sys', 'tibble',
  'utf8', 'vctrs', 'XML'

Update all/some/none? [a/s/n]: 

a

Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/data/annotation/bin/macosx/contrib/4.1/PACKAGES.rds': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/data/annotation/bin/macosx/contrib/4.1/PACKAGES.gz': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/data/experiment/bin/macosx/contrib/4.1/PACKAGES.rds': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/data/experiment/bin/macosx/contrib/4.1/PACKAGES.gz': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/workflows/bin/macosx/contrib/4.1/PACKAGES.rds': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/workflows/bin/macosx/contrib/4.1/PACKAGES.gz': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 11573Warning: cannot open URL 'https://bioconductor.org/packages/3.14/books/bin/macosx/contrib/4.1/PACKAGES.rds': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 11573Warning: cannot open URL 'https://bioconductor.org/packages/3.14/books/bin/macosx/contrib/4.1/PACKAGES.gz': HTTP status was '404 Not Found'

  There are binary versions available but the source versions are later:

no

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/askpass_1.1.tgz'
Content type 'application/x-gzip' length 21566 bytes (21 KB)
==================================================
downloaded 21 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/bit_4.0.5.tgz'
Content type 'application/x-gzip' length 1243615 bytes (1.2 MB)
==================================================
downloaded 1.2 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/bit64_4.0.5.tgz'
Content type 'application/x-gzip' length 576409 bytes (562 KB)
==================================================
downloaded 562 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/bitops_1.0-7.tgz'
Content type 'application/x-gzip' length 29174 bytes (28 KB)
==================================================
downloaded 28 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/cachem_1.0.8.tgz'
Content type 'application/x-gzip' length 67489 bytes (65 KB)
==================================================
downloaded 65 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/class_7.3-22.tgz'
Content type 'application/x-gzip' length 96138 bytes (93 KB)
==================================================
downloaded 93 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/cli_3.6.1.tgz'
Content type 'application/x-gzip' length 1365687 bytes (1.3 MB)
==================================================
downloaded 1.3 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/cluster_2.1.4.tgz'
Content type 'application/x-gzip' length 616213 bytes (601 KB)
==================================================
downloaded 601 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/curl_5.0.0.tgz'
Content type 'application/x-gzip' length 759005 bytes (741 KB)
==================================================
downloaded 741 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/farver_2.1.1.tgz'
Content type 'application/x-gzip' length 1943005 bytes (1.9 MB)
==================================================
downloaded 1.9 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/fastmap_1.1.1.tgz'
Content type 'application/x-gzip' length 201256 bytes (196 KB)
==================================================
downloaded 196 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/foreign_0.8-84.tgz'
Content type 'application/x-gzip' length 331923 bytes (324 KB)
==================================================
downloaded 324 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/glue_1.6.2.tgz'
Content type 'application/x-gzip' length 151977 bytes (148 KB)
==================================================
downloaded 148 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/jsonlite_1.8.4.tgz'
Content type 'application/x-gzip' length 1113981 bytes (1.1 MB)
==================================================
downloaded 1.1 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/KernSmooth_2.23-21.tgz'
Content type 'application/x-gzip' length 100407 bytes (98 KB)
==================================================
downloaded 98 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/lattice_0.21-8.tgz'
Content type 'application/x-gzip' length 1349836 bytes (1.3 MB)
==================================================
downloaded 1.3 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/locfit_1.5-9.7.tgz'
Content type 'application/x-gzip' length 772384 bytes (754 KB)
==================================================
downloaded 754 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/matrixStats_0.63.0.tgz'
Content type 'application/x-gzip' length 592172 bytes (578 KB)
==================================================
downloaded 578 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/mgcv_1.8-42.tgz'
Content type 'application/x-gzip' length 3737078 bytes (3.6 MB)
==================================================
downloaded 3.6 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/mime_0.12.tgz'
Content type 'application/x-gzip' length 34812 bytes (33 KB)
==================================================
downloaded 33 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/nlme_3.1-162.tgz'
Content type 'application/x-gzip' length 2400717 bytes (2.3 MB)
==================================================
downloaded 2.3 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/nnet_7.3-19.tgz'
Content type 'application/x-gzip' length 129034 bytes (126 KB)
==================================================
downloaded 126 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/openssl_2.0.6.tgz'
Content type 'application/x-gzip' length 2886168 bytes (2.8 MB)
==================================================
downloaded 2.8 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/Rcpp_1.0.10.tgz'
Content type 'application/x-gzip' length 3242867 bytes (3.1 MB)
==================================================
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#dependencies

library(DESeq2)

Loading required package: S4Vectors
Loading required package: stats4
Loading required package: BiocGenerics

Attaching package: ‘BiocGenerics’

The following objects are masked from ‘package:stats’:

    IQR, mad, sd, var, xtabs

The following objects are masked from ‘package:base’:

    anyDuplicated, append, as.data.frame, basename, cbind, colnames, dirname, do.call, duplicated, eval,
    evalq, Filter, Find, get, grep, grepl, intersect, is.unsorted, lapply, Map, mapply, match, mget, order,
    paste, pmax, pmax.int, pmin, pmin.int, Position, rank, rbind, Reduce, rownames, sapply, setdiff, sort,
    table, tapply, union, unique, unsplit, which.max, which.min


Attaching package: ‘S4Vectors’

The following objects are masked from ‘package:base’:

    expand.grid, I, unname

Loading required package: IRanges
Loading required package: GenomicRanges
Loading required package: GenomeInfoDb
Loading required package: SummarizedExperiment
Loading required package: MatrixGenerics
Loading required package: matrixStats

Attaching package: ‘MatrixGenerics’

The following objects are masked from ‘package:matrixStats’:

    colAlls, colAnyNAs, colAnys, colAvgsPerRowSet, colCollapse, colCounts, colCummaxs, colCummins,
    colCumprods, colCumsums, colDiffs, colIQRDiffs, colIQRs, colLogSumExps, colMadDiffs, colMads, colMaxs,
    colMeans2, colMedians, colMins, colOrderStats, colProds, colQuantiles, colRanges, colRanks, colSdDiffs,
    colSds, colSums2, colTabulates, colVarDiffs, colVars, colWeightedMads, colWeightedMeans,
    colWeightedMedians, colWeightedSds, colWeightedVars, rowAlls, rowAnyNAs, rowAnys, rowAvgsPerColSet,
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    rowProds, rowQuantiles, rowRanges, rowRanks, rowSdDiffs, rowSds, rowSums2, rowTabulates, rowVarDiffs,
    rowVars, rowWeightedMads, rowWeightedMeans, rowWeightedMedians, rowWeightedSds, rowWeightedVars

Loading required package: Biobase
Welcome to Bioconductor

    Vignettes contain introductory material; view with 'browseVignettes()'. To cite Bioconductor, see
    'citation("Biobase")', and for packages 'citation("pkgname")'.


Attaching package: ‘Biobase’

The following object is masked from ‘package:MatrixGenerics’:

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library(pheatmap)

Preprocessing

Reading and rounding the values of raw counts file (geneid x sample names)

## Loading Input files and creating DESeq2 object
raw_counts <- read.delim("counts.txt", header = T, row.names = 1)
raw_counts <- ceiling(raw_counts) #round them up to integers
head(raw_counts)

Loading metadata

meta_data <- read.delim("sampleGroups.txt", header = T, row.names = 1)
head(meta_data)

If the row names of metadata and col names of raw counts data: idx <- match(rownames(metadata), colnames(rawcounts)) and apply the idx to the desired data to be reordered.

Modelling DeSeq2 Object

dds <- DESeqDataSetFromMatrix(countData = raw_counts,
                              colData = meta_data,
                              design = ~group) #group is the condition:resistant or sensitive in this case

converting counts to integer mode
Warning: some variables in design formula are characters, converting to factors

dds

class: DESeqDataSet 
dim: 60313 6 
metadata(1): version
assays(1): counts
rownames(60313): ENSG00000000003 ENSG00000000005 ... __not_aligned __alignment_not_unique
rowData names(0):
colnames(6): Ovcar4_sens_1 Ovcar4_sens_2 ... Ovcar4_res_2 Ovcar4_res_3
colData names(1): group

Filter out low gene counts

keep <- rowSums(counts(dds) >= 10) >= 3
dds <- dds[keep,]

counts(dds) -> gets the count matrix rowSums() -> for each gene counts the samples that are over 10

DE Analysis

dds <- DESeq(dds)

estimating size factors
estimating dispersions
gene-wise dispersion estimates
mean-dispersion relationship
final dispersion estimates
fitting model and testing

Normalization and Log Transformation (transformed counts)

#estimateSizeFactors(dds)
#sizeFactors(dds)

#normalization
norm.counts <- counts(dds, normalized = TRUE) #normalize the counts for accuracy
write.csv(norm.counts, file = "normalized_counts.txt", row.names = TRUE)
head(norm.counts)

                Ovcar4_sens_1 Ovcar4_sens_2 Ovcar4_sens_3 Ovcar4_res_1 Ovcar4_res_2 Ovcar4_res_3
ENSG00000000003     1713.9751     1991.9480     2010.5017    1725.0727    1784.2223    1574.2618
ENSG00000000419     3320.7658     3089.6196     3120.6910    4280.8087    3391.3424    4674.2667
ENSG00000000457      324.4762      309.5147      340.3142     378.5549     431.2054     492.9857
ENSG00000000460     1027.9953     1204.8964     1407.3512    1197.4496     961.4120     966.9104
ENSG00000000971      855.5259     1627.1628     1416.1778    1721.1499     403.7051     610.8171
ENSG00000001036     2634.7860     2977.9733     2676.4191    3102.9733    2118.6265    2288.1813

#log transformation, transformed counts
vsd <- vst(dds, blind = FALSE) 

#creating as a matrix
vsd_matrix <- assay(vsd)
head(vsd_matrix)

                Ovcar4_sens_1 Ovcar4_sens_2 Ovcar4_sens_3 Ovcar4_res_1 Ovcar4_res_2 Ovcar4_res_3
ENSG00000000003     10.801797     11.010654     11.023574    10.810742    10.857502    10.684166
ENSG00000000419     11.728024     11.626197     11.640310    12.087585    11.757735    12.212452
ENSG00000000457      8.618568      8.562078      8.676013     8.805835     8.967180     9.135834
ENSG00000000460     10.101553     10.317121     10.529758    10.308668    10.011254    10.018929
ENSG00000000971      9.854852     10.729842     10.538351    10.807586     8.885186     9.411042
ENSG00000001036     11.402035     11.574313     11.424065    11.632279    11.096646    11.204261

Adding correlation to log transformation

vsd_cor <- cor(vsd_matrix) #applies correlation for a pheatmap
head(vsd_cor)

              Ovcar4_sens_1 Ovcar4_sens_2 Ovcar4_sens_3 Ovcar4_res_1 Ovcar4_res_2 Ovcar4_res_3
Ovcar4_sens_1     1.0000000     0.9915548     0.9882826    0.9635885    0.9558955    0.9657405
Ovcar4_sens_2     0.9915548     1.0000000     0.9955243    0.9661423    0.9481214    0.9572114
Ovcar4_sens_3     0.9882826     0.9955243     1.0000000    0.9677040    0.9477967    0.9572125
Ovcar4_res_1      0.9635885     0.9661423     0.9677040    1.0000000    0.9764804    0.9828277
Ovcar4_res_2      0.9558955     0.9481214     0.9477967    0.9764804    1.0000000    0.9845953
Ovcar4_res_3      0.9657405     0.9572114     0.9572125    0.9828277    0.9845953    1.0000000

vsd_cor <- data.frame(vsd_cor)

pheatmap(vsd_cor, annotation = select(meta_data, group))

1 indicates strong correlation - shows a good biological relevance. - Same groups have correlation of 1.

This plot supports that: - Chemo-sensitive and chemo-resistant samples have distinct gene expression profiles -> distinct transcriptomic level. - The clustering and correlation structure suggest clear biological separation, which is a good foundation for further differential expression or biomarker analysis.

Unsupervised Clustering

plotPCA(vsd, intgroup = c("group")) # does not want the matrix version

• PC1 explains 87% of the variance — this is the main axis separating your samples.
• Clear separation between two group -> This suggests strong transcriptomic differences between the two groups.
• PC2 (8% variance) captures minor variation within groups.

Hierarchical Clustering

#plotting hierarchical clustering using vst transformed counts
sampleDists <- dist(t(assay(vsd)))
plot(hclust(sampleDists, method = "complete"))

Plot dispersion estimates – used DESeq2 object

mean.counts <- apply(raw_counts[,1:3],1,mean)

var.counts <- apply(raw_counts[,1:3],1,var)

plotDispEsts(dds)

Key observation:

• Low-count genes (left) have high raw dispersion, but shrinkage pulls them toward the red trend line.
• High-count genes (right) show less variability and need minimal shrinkage. This confirms DESeq2’s dispersion estimation and shrinkage worked correctly, improving statistical robustness. • As mean counts increase, raw dispersions are more stable; blue ≈ black (less shrinkage needed). • Overall fit looks good (no strong systematic deviations), so dispersion modeling succeeded and you can confidently proceed to differential expression testing.

Get DE results

dds.res <- results(dds, contrast = c("group","chemo_sensitive", "chemo_resistant"), alpha = 0.05)
summary(dds.res)

out of 18577 with nonzero total read count
adjusted p-value < 0.05
LFC > 0 (up)       : 2156, 12%
LFC < 0 (down)     : 1875, 10%
outliers [1]       : 8, 0.043%
low counts [2]     : 0, 0%
(mean count < 6)
[1] see 'cooksCutoff' argument of ?results
[2] see 'independentFiltering' argument of ?results

mcols(dds.res)$description

[1] "mean of normalized counts for all samples"                       
[2] "log2 fold change (MLE): group chemo_sensitive vs chemo_resistant"
[3] "standard error: group chemo sensitive vs chemo resistant"        
[4] "Wald statistic: group chemo sensitive vs chemo resistant"        
[5] "Wald test p-value: group chemo sensitive vs chemo resistant"     
[6] "BH adjusted p-values"                                            

write.csv(as.data.frame(dds.res), file="DE_sensitiveVSresistant_results.csv")

Summary of Differential Expression Results: - 18,577 genes had a nonzero total read count (i.e., were expressed in at least one sample). - 2,156 genes were upregulated in chemo_sensitive vs chemo_resistant (12%). - 1,875 genes were downregulated (10%). - This means ~22% of all expressed genes are significantly different between the two conditions. - 8 genes (0.043%) were flagged as outliers due to extreme values (checked using Cook’s distance). - 0 genes were filtered out due to low mean counts (<6), likely because of independent filtering.

plotMA(dds.res)

• LFC > 0 is up-regulated genes; LFC < 0 is down-regulated genes.
• Grey points are the genes that are not statistically significant.
• Blue points genes with p.adj < 0.05 (significantly differentially expressed)
• There is a large number of significantly differentially expressed genes, especially in genes with moderate-to-high expression (middle x-axis).
• The triangle markers suggest some genes have very large fold changes and may be biologically interesting.

Volcano Plot

BiocManager::install('EnhancedVolcano')

'getOption("repos")' replaces Bioconductor standard repositories, see 'help("repositories", package = "BiocManager")'
for details.
Replacement repositories:
    CRAN: https://cran.rstudio.com/
Bioconductor version 3.14 (BiocManager 1.30.26), R 4.1.3 (2022-03-10)
Installing package(s) 'EnhancedVolcano'
also installing the dependencies ‘fs’, ‘rappdirs’, ‘sass’, ‘evaluate’, ‘highr’, ‘xfun’, ‘bslib’, ‘fontawesome’, ‘jquerylib’, ‘tinytex’, ‘knitr’, ‘rmarkdown’, ‘yaml’, ‘stringr’, ‘vctrs’, ‘later’, ‘stringi’, ‘extrafontdb’, ‘Rttf2pt1’, ‘digest’, ‘base64enc’, ‘htmltools’, ‘htmlwidgets’, ‘tidyr’, ‘lazyeval’, ‘crosstalk’, ‘purrr’, ‘data.table’, ‘promises’, ‘beeswarm’, ‘vipor’, ‘systemfonts’, ‘textshaping’, ‘proj4’, ‘ash’, ‘maps’, ‘extrafont’, ‘plotly’, ‘Cairo’, ‘ggbeeswarm’, ‘ragg’, ‘ggrepel’, ‘ggalt’, ‘ggrastr’

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* installing *source* package ‘evaluate’ ...
** package ‘evaluate’ successfully unpacked and MD5 sums checked
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** byte-compile and prepare package for lazy loading
** help
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** building package indices
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* DONE (evaluate)
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* installing *source* package ‘bslib’ ...
** package ‘bslib’ successfully unpacked and MD5 sums checked
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Error in loadNamespace(i, c(lib.loc, .libPaths()), versionCheck = vI[[i]]) : 
  namespace ‘htmltools’ 0.5.5 is being loaded, but >= 0.5.8 is required
Calls: <Anonymous> ... withCallingHandlers -> loadNamespace -> namespaceImport -> loadNamespace
Execution halted

ERROR: lazy loading failed for package ‘bslib’
* removing ‘/Library/Frameworks/R.framework/Versions/4.1/Resources/library/bslib’
Warning: installation of package ‘bslib’ had non-zero exit status* installing *source* package ‘fontawesome’ ...
** package ‘fontawesome’ successfully unpacked and MD5 sums checked
** using staged installation
** R
** inst
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** help
*** installing help indices
*** copying figures
** building package indices
** testing if installed package can be loaded from temporary location
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* DONE (fontawesome)
* installing *source* package ‘tinytex’ ...
** package ‘tinytex’ successfully unpacked and MD5 sums checked
** using staged installation
** R
** inst
** byte-compile and prepare package for lazy loading

Error in loadNamespace(j <- i[[1L]], c(lib.loc, .libPaths()), versionCheck = vI[[j]]) : 
  namespace ‘xfun’ 0.39 is being loaded, but >= 0.48 is required
Calls: <Anonymous> ... namespaceImportFrom -> asNamespace -> loadNamespace
Execution halted

ERROR: lazy loading failed for package ‘tinytex’
* removing ‘/Library/Frameworks/R.framework/Versions/4.1/Resources/library/tinytex’
Warning: installation of package ‘tinytex’ had non-zero exit status* installing *source* package ‘stringr’ ...
** package ‘stringr’ successfully unpacked and MD5 sums checked
** using staged installation
** R
** data
*** moving datasets to lazyload DB
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** help
*** installing help indices
*** copying figures
** building package indices
** installing vignettes
** testing if installed package can be loaded from temporary location
** testing if installed package can be loaded from final location
** testing if installed package keeps a record of temporary installation path
* DONE (stringr)
* installing *source* package ‘crosstalk’ ...
** package ‘crosstalk’ successfully unpacked and MD5 sums checked
** using staged installation
** R
** inst
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** help
*** installing help indices
** building package indices
** testing if installed package can be loaded from temporary location
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* DONE (crosstalk)
* installing *source* package ‘vipor’ ...
** package ‘vipor’ successfully unpacked and MD5 sums checked
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** data
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* DONE (vipor)
* installing *source* package ‘ggrastr’ ...
** package ‘ggrastr’ successfully unpacked and MD5 sums checked
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* DONE (ggrastr)
* installing *source* package ‘knitr’ ...
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Error in loadNamespace(j <- i[[1L]], c(lib.loc, .libPaths()), versionCheck = vI[[j]]) : 
  namespace ‘xfun’ 0.39 is being loaded, but >= 0.51 is required
Calls: <Anonymous> ... namespaceImportFrom -> asNamespace -> loadNamespace
Execution halted

ERROR: lazy loading failed for package ‘knitr’
* removing ‘/Library/Frameworks/R.framework/Versions/4.1/Resources/library/knitr’
Warning: installation of package ‘knitr’ had non-zero exit statusERROR: dependencies ‘bslib’, ‘knitr’, ‘tinytex’ are not available for package ‘rmarkdown’
* removing ‘/Library/Frameworks/R.framework/Versions/4.1/Resources/library/rmarkdown’
Warning: installation of package ‘rmarkdown’ had non-zero exit statusERROR: dependencies ‘knitr’, ‘rmarkdown’ are not available for package ‘htmlwidgets’
* removing ‘/Library/Frameworks/R.framework/Versions/4.1/Resources/library/htmlwidgets’
Warning: installation of package ‘htmlwidgets’ had non-zero exit statusERROR: dependency ‘htmlwidgets’ is not available for package ‘plotly’
* removing ‘/Library/Frameworks/R.framework/Versions/4.1/Resources/library/plotly’
Warning: installation of package ‘plotly’ had non-zero exit status
The downloaded source packages are in
    ‘/private/var/folders/db/_9mqvxh50pb53z2xy8z_xhjr0000gn/T/RtmpapzpFa/downloaded_packages’
Old packages: 'askpass', 'bit', 'bit64', 'bitops', 'cachem', 'Cairo', 'class', 'cli', 'cluster', 'curl', 'data.table',
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Update all/some/none? [a/s/n]: 

a

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library(EnhancedVolcano)

Loading required package: ggplot2

Attaching package: ‘ggplot2’

The following object is masked from ‘package:dplyr’:

    vars

Loading required package: ggrepel
Registered S3 methods overwritten by 'ggalt':
  method                  from   
  grid.draw.absoluteGrob  ggplot2
  grobHeight.absoluteGrob ggplot2
  grobWidth.absoluteGrob  ggplot2
  grobX.absoluteGrob      ggplot2
  grobY.absoluteGrob      ggplot2

EnhancedVolcano(dds.res,
    lab = rownames(dds.res),
    x = 'log2FoldChange',
    y = 'padj')

• genes to the right are up-regulated due to LFC > 0
• genes to the left are down-regulated due to LFC < 0
• higher values are more statisticaly significant
• There are many red points, meaning a large number of genes show both statistically significant changes and large expression differences between the two conditions.
• The volcano plot confirms the biological distinction between chemo-sensitive and resistant samples.
• Genes near the center LFC~0 are not statistically significant.

Most significant DE genes

plotCounts(dds, gene=which.min(dds.res$padj), intgroup="group")

Visualizing expression data with a heatmap

# thresholding for significanlty differentially expressed genes with at least two-ford change in expression
de.sign <- subset(dds.res, padj < 0.05 & abs(log2FoldChange) > 1)

# creating a vector of Ensembl id for those genes
de.sign.genes <- rownames(de.sign)

# subseting the vsd matrix to include only the de.sign.genes and scaling it by row so that we plot on the heatmap
scale.dat <- t(scale(t(assay(vsd)[de.sign.genes,])))
pheatmap(scale.dat[de.sign.genes,], cluster_rows=TRUE, show_rownames=FALSE, cluster_cols=TRUE)

Annotating DE Results

BiocManager::install('AnnotationDbi')

'getOption("repos")' replaces Bioconductor standard repositories, see 'help("repositories", package = "BiocManager")'
for details.
Replacement repositories:
    CRAN: https://cran.rstudio.com/
Bioconductor version 3.14 (BiocManager 1.30.26), R 4.1.3 (2022-03-10)
Warning: package(s) not installed when version(s) same as or greater than current; use `force = TRUE` to re-install:
  'AnnotationDbi'Old packages: 'askpass', 'bit', 'bit64', 'bitops', 'cachem', 'Cairo', 'class', 'cli', 'cluster', 'curl', 'data.table',
  'digest', 'dplyr', 'farver', 'fastmap', 'foreign', 'fs', 'ggrepel', 'glue', 'htmltools', 'jsonlite', 'KernSmooth',
  'later', 'lattice', 'locfit', 'maps', 'matrixStats', 'mgcv', 'mime', 'nlme', 'nnet', 'openssl', 'proj4', 'promises',
  'purrr', 'ragg', 'Rcpp', 'RcppArmadillo', 'RCurl', 'rlang', 'rpart', 'RSQLite', 'sass', 'spatial', 'stringi',
  'survival', 'sys', 'systemfonts', 'textshaping', 'tibble', 'tidyr', 'tidyselect', 'utf8', 'vctrs', 'xfun', 'XML',
  'yaml'

Update all/some/none? [a/s/n]: 

a

Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/data/annotation/bin/macosx/contrib/4.1/PACKAGES.rds': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/data/annotation/bin/macosx/contrib/4.1/PACKAGES.gz': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/data/experiment/bin/macosx/contrib/4.1/PACKAGES.rds': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/data/experiment/bin/macosx/contrib/4.1/PACKAGES.gz': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/workflows/bin/macosx/contrib/4.1/PACKAGES.rds': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/workflows/bin/macosx/contrib/4.1/PACKAGES.gz': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 11573Warning: cannot open URL 'https://bioconductor.org/packages/3.14/books/bin/macosx/contrib/4.1/PACKAGES.rds': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 11573Warning: cannot open URL 'https://bioconductor.org/packages/3.14/books/bin/macosx/contrib/4.1/PACKAGES.gz': HTTP status was '404 Not Found'

  There are binary versions available but the source versions are later:

no

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/askpass_1.1.tgz'
Content type 'application/x-gzip' length 21566 bytes (21 KB)
==================================================
downloaded 21 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/bit_4.0.5.tgz'
Content type 'application/x-gzip' length 1243615 bytes (1.2 MB)
==================================================
downloaded 1.2 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/bit64_4.0.5.tgz'
Content type 'application/x-gzip' length 576409 bytes (562 KB)
==================================================
downloaded 562 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/bitops_1.0-7.tgz'
Content type 'application/x-gzip' length 29174 bytes (28 KB)
==================================================
downloaded 28 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/cachem_1.0.8.tgz'
Content type 'application/x-gzip' length 67489 bytes (65 KB)
==================================================
downloaded 65 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/Cairo_1.6-0.tgz'
Content type 'application/x-gzip' length 22861382 bytes (21.8 MB)
==================================================
downloaded 21.8 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/class_7.3-22.tgz'
Content type 'application/x-gzip' length 96138 bytes (93 KB)
==================================================
downloaded 93 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/cli_3.6.1.tgz'
Content type 'application/x-gzip' length 1365687 bytes (1.3 MB)
==================================================
downloaded 1.3 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/cluster_2.1.4.tgz'
Content type 'application/x-gzip' length 616213 bytes (601 KB)
==================================================
downloaded 601 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/curl_5.0.0.tgz'
Content type 'application/x-gzip' length 759005 bytes (741 KB)
==================================================
downloaded 741 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/data.table_1.14.8.tgz'
Content type 'application/x-gzip' length 2376122 bytes (2.3 MB)
==================================================
downloaded 2.3 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/digest_0.6.31.tgz'
Content type 'application/x-gzip' length 298704 bytes (291 KB)
==================================================
downloaded 291 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/dplyr_1.1.2.tgz'
Content type 'application/x-gzip' length 1561346 bytes (1.5 MB)
==================================================
downloaded 1.5 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/farver_2.1.1.tgz'
Content type 'application/x-gzip' length 1943005 bytes (1.9 MB)
==================================================
downloaded 1.9 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/fastmap_1.1.1.tgz'
Content type 'application/x-gzip' length 201256 bytes (196 KB)
==================================================
downloaded 196 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/foreign_0.8-84.tgz'
Content type 'application/x-gzip' length 331923 bytes (324 KB)
==================================================
downloaded 324 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/fs_1.6.2.tgz'
Content type 'application/x-gzip' length 569071 bytes (555 KB)
==================================================
downloaded 555 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/ggrepel_0.9.3.tgz'
Content type 'application/x-gzip' length 577454 bytes (563 KB)
==================================================
downloaded 563 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/glue_1.6.2.tgz'
Content type 'application/x-gzip' length 151977 bytes (148 KB)
==================================================
downloaded 148 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/htmltools_0.5.5.tgz'
Content type 'application/x-gzip' length 346776 bytes (338 KB)
==================================================
downloaded 338 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/jsonlite_1.8.4.tgz'
Content type 'application/x-gzip' length 1113981 bytes (1.1 MB)
==================================================
downloaded 1.1 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/KernSmooth_2.23-21.tgz'
Content type 'application/x-gzip' length 100407 bytes (98 KB)
==================================================
downloaded 98 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/later_1.3.1.tgz'
Content type 'application/x-gzip' length 612981 bytes (598 KB)
==================================================
downloaded 598 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/lattice_0.21-8.tgz'
Content type 'application/x-gzip' length 1349836 bytes (1.3 MB)
==================================================
downloaded 1.3 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/locfit_1.5-9.7.tgz'
Content type 'application/x-gzip' length 772384 bytes (754 KB)
==================================================
downloaded 754 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/maps_3.4.1.tgz'
Content type 'application/x-gzip' length 3105432 bytes (3.0 MB)
==================================================
downloaded 3.0 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/matrixStats_0.63.0.tgz'
Content type 'application/x-gzip' length 592172 bytes (578 KB)
==================================================
downloaded 578 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/mgcv_1.8-42.tgz'
Content type 'application/x-gzip' length 3737078 bytes (3.6 MB)
==================================================
downloaded 3.6 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/mime_0.12.tgz'
Content type 'application/x-gzip' length 34812 bytes (33 KB)
==================================================
downloaded 33 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/nlme_3.1-162.tgz'
Content type 'application/x-gzip' length 2400717 bytes (2.3 MB)
==================================================
downloaded 2.3 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/nnet_7.3-19.tgz'
Content type 'application/x-gzip' length 129034 bytes (126 KB)
==================================================
downloaded 126 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/openssl_2.0.6.tgz'
Content type 'application/x-gzip' length 2886168 bytes (2.8 MB)
==================================================
downloaded 2.8 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/proj4_1.0-12.tgz'
Content type 'application/x-gzip' length 17782645 bytes (17.0 MB)
==================================================
downloaded 17.0 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/promises_1.2.0.1.tgz'
Content type 'application/x-gzip' length 1782018 bytes (1.7 MB)
==================================================
downloaded 1.7 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/purrr_1.0.1.tgz'
Content type 'application/x-gzip' length 509931 bytes (497 KB)
==================================================
downloaded 497 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/ragg_1.2.5.tgz'
Content type 'application/x-gzip' length 9223518 bytes (8.8 MB)
==================================================
downloaded 8.8 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/Rcpp_1.0.10.tgz'
Content type 'application/x-gzip' length 3242867 bytes (3.1 MB)
==================================================
downloaded 3.1 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/RcppArmadillo_0.12.2.0.0.tgz'
Content type 'application/x-gzip' length 1570686 bytes (1.5 MB)
==================================================
downloaded 1.5 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/RCurl_1.98-1.12.tgz'
Content type 'application/x-gzip' length 1089009 bytes (1.0 MB)
==================================================
downloaded 1.0 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/rlang_1.1.1.tgz'
Content type 'application/x-gzip' length 1852475 bytes (1.8 MB)
==================================================
downloaded 1.8 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/rpart_4.1.19.tgz'
Content type 'application/x-gzip' length 980040 bytes (957 KB)
==================================================
downloaded 957 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/RSQLite_2.3.1.tgz'
Content type 'application/x-gzip' length 4340278 bytes (4.1 MB)
==================================================
downloaded 4.1 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/sass_0.4.6.tgz'
Content type 'application/x-gzip' length 2392703 bytes (2.3 MB)
==================================================
downloaded 2.3 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/spatial_7.3-16.tgz'
Content type 'application/x-gzip' length 157695 bytes (153 KB)
==================================================
downloaded 153 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/stringi_1.7.12.tgz'
Content type 'application/x-gzip' length 14448461 bytes (13.8 MB)
==================================================
downloaded 13.8 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/survival_3.5-5.tgz'
Content type 'application/x-gzip' length 6768208 bytes (6.5 MB)
==================================================
downloaded 6.5 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/sys_3.4.1.tgz'
Content type 'application/x-gzip' length 47312 bytes (46 KB)
==================================================
downloaded 46 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/systemfonts_1.0.4.tgz'
Content type 'application/x-gzip' length 5900454 bytes (5.6 MB)
==================================================
downloaded 5.6 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/textshaping_0.3.6.tgz'
Content type 'application/x-gzip' length 1291817 bytes (1.2 MB)
==================================================
downloaded 1.2 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/tibble_3.2.1.tgz'
Content type 'application/x-gzip' length 676416 bytes (660 KB)
==================================================
downloaded 660 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/tidyr_1.3.0.tgz'
Content type 'application/x-gzip' length 1324625 bytes (1.3 MB)
==================================================
downloaded 1.3 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/tidyselect_1.2.0.tgz'
Content type 'application/x-gzip' length 219384 bytes (214 KB)
==================================================
downloaded 214 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/utf8_1.2.3.tgz'
Content type 'application/x-gzip' length 196912 bytes (192 KB)
==================================================
downloaded 192 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/vctrs_0.6.2.tgz'
Content type 'application/x-gzip' length 1850681 bytes (1.8 MB)
==================================================
downloaded 1.8 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/xfun_0.39.tgz'
Content type 'application/x-gzip' length 430615 bytes (420 KB)
==================================================
downloaded 420 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/XML_3.99-0.14.tgz'
Content type 'application/x-gzip' length 1873852 bytes (1.8 MB)
==================================================
downloaded 1.8 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/yaml_2.3.7.tgz'
Content type 'application/x-gzip' length 204951 bytes (200 KB)
==================================================
downloaded 200 KB

The downloaded binary packages are in
    /var/folders/db/_9mqvxh50pb53z2xy8z_xhjr0000gn/T//RtmpapzpFa/downloaded_packages

BiocManager::install("org.Hs.eg.db")

'getOption("repos")' replaces Bioconductor standard repositories, see 'help("repositories", package = "BiocManager")'
for details.
Replacement repositories:
    CRAN: https://cran.rstudio.com/
Bioconductor version 3.14 (BiocManager 1.30.26), R 4.1.3 (2022-03-10)
Installing package(s) 'org.Hs.eg.db'
Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/data/annotation/bin/macosx/contrib/4.1/PACKAGES.rds': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/data/annotation/bin/macosx/contrib/4.1/PACKAGES.gz': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/data/experiment/bin/macosx/contrib/4.1/PACKAGES.rds': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/data/experiment/bin/macosx/contrib/4.1/PACKAGES.gz': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/workflows/bin/macosx/contrib/4.1/PACKAGES.rds': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/workflows/bin/macosx/contrib/4.1/PACKAGES.gz': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 11573Warning: cannot open URL 'https://bioconductor.org/packages/3.14/books/bin/macosx/contrib/4.1/PACKAGES.rds': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 11573Warning: cannot open URL 'https://bioconductor.org/packages/3.14/books/bin/macosx/contrib/4.1/PACKAGES.gz': HTTP status was '404 Not Found'installing the source package ‘org.Hs.eg.db’

trying URL 'https://bioconductor.org/packages/3.14/data/annotation/src/contrib/org.Hs.eg.db_3.14.0.tar.gz'
Content type 'application/octet-stream' length 82195112 bytes (78.4 MB)
==================================================
downloaded 78.4 MB

* installing *source* package ‘org.Hs.eg.db’ ...
** using staged installation
** R
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
** building package indices
** testing if installed package can be loaded from temporary location
** testing if installed package can be loaded from final location
** testing if installed package keeps a record of temporary installation path
* DONE (org.Hs.eg.db)

The downloaded source packages are in
    ‘/private/var/folders/db/_9mqvxh50pb53z2xy8z_xhjr0000gn/T/RtmpapzpFa/downloaded_packages’
Old packages: 'askpass', 'bit', 'bit64', 'bitops', 'cachem', 'Cairo', 'class', 'cli', 'cluster', 'curl', 'data.table',
  'digest', 'dplyr', 'farver', 'fastmap', 'foreign', 'fs', 'ggrepel', 'glue', 'htmltools', 'jsonlite', 'KernSmooth',
  'later', 'lattice', 'locfit', 'maps', 'matrixStats', 'mgcv', 'mime', 'nlme', 'nnet', 'openssl', 'proj4', 'promises',
  'purrr', 'ragg', 'Rcpp', 'RcppArmadillo', 'RCurl', 'rlang', 'rpart', 'RSQLite', 'sass', 'spatial', 'stringi',
  'survival', 'sys', 'systemfonts', 'textshaping', 'tibble', 'tidyr', 'tidyselect', 'utf8', 'vctrs', 'xfun', 'XML',
  'yaml'

Update all/some/none? [a/s/n]: 

a

Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/data/annotation/bin/macosx/contrib/4.1/PACKAGES.rds': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/data/annotation/bin/macosx/contrib/4.1/PACKAGES.gz': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/data/experiment/bin/macosx/contrib/4.1/PACKAGES.rds': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/data/experiment/bin/macosx/contrib/4.1/PACKAGES.gz': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/workflows/bin/macosx/contrib/4.1/PACKAGES.rds': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.14/workflows/bin/macosx/contrib/4.1/PACKAGES.gz': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 11573Warning: cannot open URL 'https://bioconductor.org/packages/3.14/books/bin/macosx/contrib/4.1/PACKAGES.rds': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 11573Warning: cannot open URL 'https://bioconductor.org/packages/3.14/books/bin/macosx/contrib/4.1/PACKAGES.gz': HTTP status was '404 Not Found'

  There are binary versions available but the source versions are later:

no

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/askpass_1.1.tgz'
Content type 'application/x-gzip' length 21566 bytes (21 KB)
==================================================
downloaded 21 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/bit_4.0.5.tgz'
Content type 'application/x-gzip' length 1243615 bytes (1.2 MB)
==================================================
downloaded 1.2 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/bit64_4.0.5.tgz'
Content type 'application/x-gzip' length 576409 bytes (562 KB)
==================================================
downloaded 562 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/bitops_1.0-7.tgz'
Content type 'application/x-gzip' length 29174 bytes (28 KB)
==================================================
downloaded 28 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/cachem_1.0.8.tgz'
Content type 'application/x-gzip' length 67489 bytes (65 KB)
==================================================
downloaded 65 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/Cairo_1.6-0.tgz'
Content type 'application/x-gzip' length 22861382 bytes (21.8 MB)
==================================================
downloaded 21.8 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/class_7.3-22.tgz'
Content type 'application/x-gzip' length 96138 bytes (93 KB)
==================================================
downloaded 93 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/cli_3.6.1.tgz'
Content type 'application/x-gzip' length 1365687 bytes (1.3 MB)
==================================================
downloaded 1.3 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/cluster_2.1.4.tgz'
Content type 'application/x-gzip' length 616213 bytes (601 KB)
==================================================
downloaded 601 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/curl_5.0.0.tgz'
Content type 'application/x-gzip' length 759005 bytes (741 KB)
==================================================
downloaded 741 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/data.table_1.14.8.tgz'
Content type 'application/x-gzip' length 2376122 bytes (2.3 MB)
==================================================
downloaded 2.3 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/digest_0.6.31.tgz'
Content type 'application/x-gzip' length 298704 bytes (291 KB)
==================================================
downloaded 291 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/dplyr_1.1.2.tgz'
Content type 'application/x-gzip' length 1561346 bytes (1.5 MB)
==================================================
downloaded 1.5 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/farver_2.1.1.tgz'
Content type 'application/x-gzip' length 1943005 bytes (1.9 MB)
==================================================
downloaded 1.9 MB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/fastmap_1.1.1.tgz'
Content type 'application/x-gzip' length 201256 bytes (196 KB)
==================================================
downloaded 196 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/foreign_0.8-84.tgz'
Content type 'application/x-gzip' length 331923 bytes (324 KB)
==================================================
downloaded 324 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/fs_1.6.2.tgz'
Content type 'application/x-gzip' length 569071 bytes (555 KB)
==================================================
downloaded 555 KB

trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.1/ggrepel_0.9.3.tgz'
Content type 'application/x-gzip' length 577454 bytes (563 KB)
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The downloaded binary packages are in
    /var/folders/db/_9mqvxh50pb53z2xy8z_xhjr0000gn/T//RtmpapzpFa/downloaded_packages

library(AnnotationDbi)
library(org.Hs.eg.db)

res.df <- as.data.frame(dds.res) # putting the de.sign into a data.frame to help the next step to work
res.annot <- mapIds(org.Hs.eg.db, keys = rownames(res.df), keytype = "ENSEMBL", column = "SYMBOL") #mapping ensembl ids to Gene Symbols

'select()' returned 1:many mapping between keys and columns

res.df.annot <- cbind(res.df,gene.symbol = res.annot) #binding the annotations to the sign.df

head(res.df.annot)

write.csv(res.df.annot, file = "DE_sensitiveVSresistant_results_wGeneSymbol.csv")

EnhancedVolcano(res.df.annot,
    lab = res.df.annot$gene.symbol,
    x = 'log2FoldChange',
    y = 'padj')

Over-representation test

Install R packages and load libraries

BiocManager::install("clusterProfiler")

'getOption("repos")' replaces Bioconductor standard repositories, see 'help("repositories", package = "BiocManager")'
for details.
Replacement repositories:
    CRAN: https://cran.rstudio.com/
Bioconductor version 3.15 (BiocManager 1.30.26), R 4.2.3 (2023-03-15)
Installing package(s) 'clusterProfiler'
also installing the dependencies ‘gridGraphics’, ‘tweenr’, ‘polyclip’, ‘RcppArmadillo’, ‘gridExtra’, ‘fastmatch’, ‘ggfun’, ‘ggplotify’, ‘patchwork’, ‘ggforce’, ‘ggrepel’, ‘viridis’, ‘tidygraph’, ‘graphlayouts’, ‘S7’, ‘ape’, ‘tidytree’, ‘treeio’, ‘DO.db’, ‘fgsea’, ‘reshape2’, ‘aplot’, ‘ggraph’, ‘igraph’, ‘scatterpie’, ‘shadowtext’, ‘ggtree’, ‘downloader’, ‘DOSE’, ‘enrichplot’, ‘GOSemSim’, ‘plyr’, ‘qvalue’, ‘yulab.utils’

Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.15/data/annotation/bin/macosx/contrib/4.2/PACKAGES.rds': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.15/data/annotation/bin/macosx/contrib/4.2/PACKAGES.gz': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.15/data/experiment/bin/macosx/contrib/4.2/PACKAGES.rds': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.15/data/experiment/bin/macosx/contrib/4.2/PACKAGES.gz': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.15/workflows/bin/macosx/contrib/4.2/PACKAGES.rds': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 250Warning: cannot open URL 'https://mghp.osn.xsede.org/bir190004-bucket01/archive.bioconductor.org/packages/3.15/workflows/bin/macosx/contrib/4.2/PACKAGES.gz': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 11573Warning: cannot open URL 'https://bioconductor.org/packages/3.15/books/bin/macosx/contrib/4.2/PACKAGES.rds': HTTP status was '404 Not Found'Warning: downloaded length 0 != reported length 11573Warning: cannot open URL 'https://bioconductor.org/packages/3.15/books/bin/macosx/contrib/4.2/PACKAGES.gz': HTTP status was '404 Not Found'

  There are binary versions available but the source versions are later:

no

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The downloaded binary packages are in
    /var/folders/db/_9mqvxh50pb53z2xy8z_xhjr0000gn/T//Rtmp173bOF/downloaded_packages

installing the source packages ‘ggfun’, ‘patchwork’, ‘viridis’, ‘DO.db’, ‘aplot’, ‘scatterpie’, ‘shadowtext’, ‘downloader’, ‘yulab.utils’

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* installing *source* package ‘patchwork’ ...
** package ‘patchwork’ successfully unpacked and MD5 sums checked
** using staged installation
** R
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
*** copying figures
** building package indices
** installing vignettes
** testing if installed package can be loaded from temporary location
** testing if installed package can be loaded from final location
** testing if installed package keeps a record of temporary installation path
* DONE (patchwork)
* installing *source* package ‘viridis’ ...
** package ‘viridis’ successfully unpacked and MD5 sums checked
** using staged installation
** R
** data
*** moving datasets to lazyload DB
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
*** copying figures
** building package indices
** installing vignettes
** testing if installed package can be loaded from temporary location
** testing if installed package can be loaded from final location
** testing if installed package keeps a record of temporary installation path
* DONE (viridis)
* installing *source* package ‘DO.db’ ...
** using staged installation
** R
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
** building package indices
** testing if installed package can be loaded from temporary location
** testing if installed package can be loaded from final location
** testing if installed package keeps a record of temporary installation path
* DONE (DO.db)
* installing *source* package ‘shadowtext’ ...
** package ‘shadowtext’ successfully unpacked and MD5 sums checked
** using staged installation
** R
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
** building package indices
** installing vignettes
** testing if installed package can be loaded from temporary location
** testing if installed package can be loaded from final location
** testing if installed package keeps a record of temporary installation path
* DONE (shadowtext)
* installing *source* package ‘downloader’ ...
** package ‘downloader’ successfully unpacked and MD5 sums checked
** using staged installation
** R
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
** building package indices
** testing if installed package can be loaded from temporary location
** testing if installed package can be loaded from final location
** testing if installed package keeps a record of temporary installation path
* DONE (downloader)
* installing *source* package ‘yulab.utils’ ...
** package ‘yulab.utils’ successfully unpacked and MD5 sums checked
** using staged installation
** R
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
** building package indices
** testing if installed package can be loaded from temporary location
** testing if installed package can be loaded from final location
** testing if installed package keeps a record of temporary installation path
* DONE (yulab.utils)
* installing *source* package ‘ggfun’ ...
** package ‘ggfun’ successfully unpacked and MD5 sums checked
** using staged installation
** R
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
** building package indices
** installing vignettes
** testing if installed package can be loaded from temporary location
** testing if installed package can be loaded from final location
** testing if installed package keeps a record of temporary installation path
* DONE (ggfun)
* installing *source* package ‘aplot’ ...
** package ‘aplot’ successfully unpacked and MD5 sums checked
** using staged installation
** R
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
*** copying figures
** building package indices
** testing if installed package can be loaded from temporary location
** testing if installed package can be loaded from final location
** testing if installed package keeps a record of temporary installation path
* DONE (aplot)
* installing *source* package ‘scatterpie’ ...
** package ‘scatterpie’ successfully unpacked and MD5 sums checked
** using staged installation
** R
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
** building package indices
** installing vignettes
** testing if installed package can be loaded from temporary location
** testing if installed package can be loaded from final location
** testing if installed package keeps a record of temporary installation path
* DONE (scatterpie)

The downloaded source packages are in
    ‘/private/var/folders/db/_9mqvxh50pb53z2xy8z_xhjr0000gn/T/Rtmp173bOF/downloaded_packages’
Old packages: 'ape', 'brio', 'curl', 'data.table', 'diffobj', 'fastmatch', 'gert', 'ggforce', 'ggraph', 'ggrepel',
  'graphlayouts', 'httr2', 'igraph', 'KernSmooth', 'mclust', 'nlme', 'openssl', 'polyclip', 'processx', 'profvis', 'ps',
  'purrr', 'ragg', 'RcppArmadillo', 'RCurl', 'restfulr', 'roxygen2', 'rprojroot', 'S7', 'systemfonts', 'testthat',
  'textshaping', 'tidygraph', 'tweenr', 'waldo', 'XML', 'zip'

Update all/some/none? [a/s/n]: 

a

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  There are binary versions available but the source versions are later:

no

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The downloaded binary packages are in
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installing the source packages ‘httr2’, ‘rprojroot’, ‘waldo’

trying URL 'https://cran.rstudio.com/src/contrib/httr2_1.2.0.tar.gz'
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* installing *source* package ‘httr2’ ...
** package ‘httr2’ successfully unpacked and MD5 sums checked
** using staged installation
** R
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
*** copying figures
** building package indices
** installing vignettes
** testing if installed package can be loaded from temporary location
** testing if installed package can be loaded from final location
** testing if installed package keeps a record of temporary installation path
* DONE (httr2)
* installing *source* package ‘rprojroot’ ...
** package ‘rprojroot’ successfully unpacked and MD5 sums checked
** using staged installation
** R
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
*** copying figures
** building package indices
** installing vignettes
** testing if installed package can be loaded from temporary location
** testing if installed package can be loaded from final location
** testing if installed package keeps a record of temporary installation path
* DONE (rprojroot)
* installing *source* package ‘waldo’ ...
** package ‘waldo’ successfully unpacked and MD5 sums checked
** using staged installation
** R
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
*** copying figures
** building package indices
** testing if installed package can be loaded from temporary location
** testing if installed package can be loaded from final location
** testing if installed package keeps a record of temporary installation path
* DONE (waldo)

The downloaded source packages are in
    ‘/private/var/folders/db/_9mqvxh50pb53z2xy8z_xhjr0000gn/T/Rtmp173bOF/downloaded_packages’

library(clusterProfiler)

Registered S3 method overwritten by 'data.table':
  method           from
  print.data.table     
Registered S3 methods overwritten by 'treeio':
  method              from    
  MRCA.phylo          tidytree
  MRCA.treedata       tidytree
  Nnode.treedata      tidytree
  Ntip.treedata       tidytree
  ancestor.phylo      tidytree
  ancestor.treedata   tidytree
  child.phylo         tidytree
  child.treedata      tidytree
  full_join.phylo     tidytree
  full_join.treedata  tidytree
  groupClade.phylo    tidytree
  groupClade.treedata tidytree
  groupOTU.phylo      tidytree
  groupOTU.treedata   tidytree
  is.rooted.treedata  tidytree
  nodeid.phylo        tidytree
  nodeid.treedata     tidytree
  nodelab.phylo       tidytree
  nodelab.treedata    tidytree
  offspring.phylo     tidytree
  offspring.treedata  tidytree
  parent.phylo        tidytree
  parent.treedata     tidytree
  root.treedata       tidytree
  rootnode.phylo      tidytree
  sibling.phylo       tidytree
clusterProfiler v4.4.4  For help: https://yulab-smu.top/biomedical-knowledge-mining-book/

If you use clusterProfiler in published research, please cite:
T Wu, E Hu, S Xu, M Chen, P Guo, Z Dai, T Feng, L Zhou, W Tang, L Zhan, X Fu, S Liu, X Bo, and G Yu. clusterProfiler 4.0: A universal enrichment tool for interpreting omics data. The Innovation. 2021, 2(3):100141

Attaching package: ‘clusterProfiler’

The following object is masked from ‘package:stats’:

    filter

install.packages("msigdbr")

also installing the dependencies ‘assertthat’, ‘babelgene’

  There is a binary version available but the source version is later:

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trying URL 'https://cran.rstudio.com/bin/macosx/contrib/4.2/babelgene_22.9.tgz'
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The downloaded binary packages are in
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installing the source package ‘msigdbr’

trying URL 'https://cran.rstudio.com/src/contrib/msigdbr_25.1.0.tar.gz'
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* installing *source* package ‘msigdbr’ ...
** package ‘msigdbr’ successfully unpacked and MD5 sums checked
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** byte-compile and prepare package for lazy loading
** help
*** installing help indices
*** copying figures
** building package indices
** testing if installed package can be loaded from temporary location
** testing if installed package can be loaded from final location
** testing if installed package keeps a record of temporary installation path
* DONE (msigdbr)

The downloaded source packages are in
    ‘/private/var/folders/db/_9mqvxh50pb53z2xy8z_xhjr0000gn/T/Rtmp173bOF/downloaded_packages’

library(msigdbr)

msigdbr is an R package that gives you easy access to gene sets from the Molecular Signatures Database (MSigDB) — a widely used resource for gene set enrichment analysis (GSEA).

Load the C5 collection from MSigDB i.e. Gene Ontologies

go_gene_sets <- msigdbr(species = "human", category = "C5")  #Load C5

Warning: The `category` argument of `msigdbr()` is deprecated as of msigdbr 10.0.0.
Please use the `collection` argument instead.
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msigdbr_t2g <- go_gene_sets %>% dplyr::distinct(gs_name,gene_symbol) %>% as.data.frame() # make a dataframe of the genesets and the corresponding Ensemble gene ids

Load the DE results file and subset for significantly upregulated genes

DE.res <- read.csv("DE_sensitiveVSresistant_results_wGeneSymbol.csv", header = TRUE, row.names = 1)
DE.res.sign <- subset(DE.res, padj < 0.05 & log2FoldChange > 1)

Perform enrichment analysis and write results in a .csv file

enrichRes <- enricher(gene = DE.res.sign$gene.symbol, TERM2GENE = msigdbr_t2g, pvalueCutoff = 0.05, pAdjustMethod = "BH")
write.csv(enrichRes@result, file = "GO_sensitiveVSresistant_up.csv", row.names = FALSE)
head(enrichRes)

Pre-ranked GSEA

Installing packages

#BiocManager::install("fgsea")
library(fgsea)

#install.packages("data.table")
library(data.table)

BiocManager::install("qusage")

'getOption("repos")' replaces Bioconductor standard repositories, see 'help("repositories", package = "BiocManager")'
for details.
Replacement repositories:
    CRAN: https://cran.rstudio.com/
Bioconductor version 3.15 (BiocManager 1.30.26), R 4.2.3 (2023-03-15)
Installing package(s) 'qusage'
also installing the dependencies ‘estimability’, ‘mvtnorm’, ‘emmeans’, ‘fftw’

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* installing *source* package ‘estimability’ ...
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** R
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** help
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** testing if installed package keeps a record of temporary installation path
* DONE (estimability)
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* DONE (emmeans)

The downloaded source packages are in
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Old packages: 'ape', 'brio', 'curl', 'data.table', 'diffobj', 'fastmatch', 'fftw', 'gert', 'ggforce', 'ggraph',
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Update all/some/none? [a/s/n]: 

a

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library(qusage)

Loading required package: limma

Attaching package: ‘qusage’

The following object is masked from ‘package:clusterProfiler’:

    read.gmt

#install.packages("ggplot2")
library(ggplot2)

Load the Gene Set collection (Note you need to download the gmt file of the collection of your interest from MSigDB)

gmt.file <- read.gmt("c5.go.bp.v7.4.symbols.gmt")

Ranking our DE results

DE.res.ranked <- DE.res[order(DE.res$log2FoldChange, decreasing = T), ]

DE.ranks <- setNames(DE.res.ranked$log2FoldChange, DE.res.ranked$gene.symbol) # creating a named vector of the ranks

Running fgsea

fgseaRes <- fgsea(gmt.file, DE.ranks, minSize=15, maxSize=500)

Warning: There are duplicate gene names, fgsea may produce unexpected results.

# writing the results into a file
fwrite(fgseaRes, file = "PrerankedGSEA_gobp_sensitiveVSresistant.txt", sep = "\t", sep2=c("", " ", ""))

Producing an enrichment plot

topPathways <- fgseaRes[order(padj)][padj <= 0.05]$pathway
EnPlot <- plotEnrichment(gmt.file[["GOBP_EPITHELIUM_DEVELOPMENT"]],DE.ranks) + labs(title="GOBP_EPITHELIUM_DEVELOPMENT") + theme(text = element_text(size=20))
EnPlot2 <- plotEnrichment(gmt.file[["GOBP_OXIDATIVE_PHOSPHORYLATION"]],DE.ranks) + labs(title="GOBP_OXIDATIVE_PHOSPHORYLATION") + theme(text = element_text(size=20))

EnPlot

EnPlot2

X-axis (“rank”): Genes ranked from most upregulated (left) to most downregulated (right) based on a metric (e.g., log2FC, signal-to-noise).

Black ticks: Locations of genes from the epithelium development gene set along the ranked gene list.

Green curve: The enrichment score (ES) — it reflects how concentrated the gene set is near the top (left) or bottom (right) of the ranked list.

If the peak is on the left: gene set is enriched in upregulated genes.

If on the right: enriched in downregulated genes.

Red dashed lines: Indicate the range of observed ES scores; useful for permutation testing.

✅ Interpretation of your plot: The green line peaks early, around the left side of the ranked gene list.

This indicates that genes in the GOBP_EPITHELIUM_DEVELOPMENT gene set are enriched at the top of the ranking.

Therefore, this gene set is upregulated (i.e., activated or associated) in your chemo-sensitive group (if that’s the direction of the ranking).

🧠 Biological interpretation: Epithelium development–related genes are significantly enriched among the most upregulated genes in your condition (likely chemo-sensitive).

This may suggest that epithelial differentiation or maintenance is more active in chemo-sensitive samples.